Undesirable and dangerous side effects and adverse drug interactions are well known for the predominantly synthetic organic pharmaceuticals that have been widely administered over the past several decades. These adverse effects have led many research groups to go back and study, in greater detail, the medicinal properties and mechanisms of action of many natural compounds. Ancient cultures have long been aware of the medicinal properties of the natural product, honey. The subject matter of the present invention involves novel medicinal activities associated with methylglyoxal-fortified buckwheat honey.
In one embodiment of the present invention, various antibacterial mechanisms are combined into a honey. Previously, different antibacterial mechanisms have been known to exist only separately in honeys derived from different floral sources. Honey has been widely accepted as both food and medicine by most, if not all, generations, traditions, and civilizations, both ancient and modern. Although honey has been used by humans for more than 5,000 years to treat a variety of ailments, it has been recognized for almost as long that honeys derived from some floral sources are more medicinal than others. As a general rule, darker colored honeys have more medicinal activities than light-colored honeys. Many studies have shown that medicinal honey influences biological systems as antioxidant anti-inflammatory, and antimicrobial. In addition honey acts as an autolytic debridement agent on wounds, as a cough suppressant, analgesic, remedy for dyspepsia, and natural anticancer agent.
One of the darkest honeys is buckwheat honey, which has been shown to have one of the highest antioxidant, anti-inflammatory, and antibacterial activities of any honey variety tested. Because of the bacterial resistance problems that have arisen from the overuse and misuse of antibiotics, the antibacterial activity of honey is the activity that has renewed the interest in honey, particularly for treatment of hard-to-heal (chronic) wounds. But the antibacterial activity of honeys derived from different floral sources has been found to be due to different mechanisms. Early on, honey's antibacterial activity was attributed to its osmotic effect and to its low pH, but these have subsequently been found to contribute only minor antibacterial effects. The first factor discovered that contributes a major antibacterial activity in honey was hydrogen peroxide, but it's generation and concentration are under the control of a number of important effects.
First, hydrogen peroxide is not a constituent of the nectar from which honey is produced. It is derived from the enzymatic activity of glucose oxidase acting on glucose. The maturation of honey from plant nectar is dependent upon the activities of several enzymes, most of which are derived from the hypopharyngeal gland of the honey bee. Diastase (amylase), derived from the bee, breaks down starch to smaller carbohydrates (dextrins, oligo-, di- and monosaccharides [glucose]). Invertase, derived from the bee, converts sucrose, the primary sugar in nectar, into glucose and fructose. Glucose oxidase, also derived from the bee, catalyzes the oxidation of glucose by molecular oxygen to gluconolactone, which subsequently hydrolyzes spontaneously to gluconic acid and hydrogen peroxide. Gluconic acid is the primary acid in honey responsible for most of honey's acidity and low pH, and hydrogen peroxide is the primary antibacterial agent in most medicinal honeys.
Second, the production of hydrogen peroxide is very slow in mature honey for two reasons: i) the activity of glucose oxidase is depressed by high osmotic pressure, and ii) the spontaneous conversion of gluconolactone to gluconic acid and hydrogen peroxide is a hydrolysis reaction requiring water, which is unavailable in ripe honey. Most hydrogen peroxide present in ripe honey was generated while water was available as the honey was being ripened and dried by the bees. And when ripened honey is subsequently diluted, by wound fluid for example, this reaction speeds up again. Upon dilution of medicinal honey, the rate of hydrogen peroxide generation is continuous and can reach concentrations in excess of 4 mmol/L, with a mean of about 1-2 mmol/L. This relatively low concentration is nevertheless high enough to provide a substantial antibacterial activity, and yet is about 1000-times less then the 3% solution commonly used as an antiseptic; which high concentration has been associated with tissue damage, including damage to fibroblast cells from human skin. Furthermore, the continuous production of hydrogen peroxide in diluted honey produces a long-lasting antiseptic effect that is most sought after in fighting infections and wounds. It has been reported that hydrogen peroxide is more effective when supplied by continuous generation from glucose oxidase catalysis, as in honey, than when added as a single bolus.
Third, in addition to the glucose/glucose oxidase system as a main source of hydrogen peroxide generation, plant-derived polyphenols present in some honeys provide a supplementary source of hydrogen peroxide. Honeys with high concentrations of polyphenols, such as buckwheat honey, have higher hydrogen peroxide levels due to this second method of hydrogen peroxide generation. The mechanism of this action is likely from the auto-oxidation of polyphenols yielding both hydrogen peroxide and phenoxyl-radicals. Furthermore, redox-active phenolics appear to be active intermediates that confer additional oxidative activity on hydrogen peroxide. In addition, the chemical interaction of honey phenolics with hydrogen peroxide results in products that degrade bacterial DNA. In the presence of transition metal ions, via the Fenton reaction, hydrogen peroxide is also converted to hydroxyl radicals. Both the phenoxyl- and hydroxyl-radicals have been shown to induce strand breaks in DNA. Thus, a second factor present in some honeys that contribute to its antibacterial effect are polyphenols.
A third factor found in honey that has antibacterial activity is methylglyoxal (MGO), but this agent has only been found in honey derived from certain floral species of the Leptospermum genus of shrubs and small trees found in New Zealand, Australia, Malaysia, and Indonesia. Originally referred to as UMF (Unique Manuka Factor), methylglyoxal has been found to originate in honey from dihydroxyacetone present in the nectar of Leptospermum flowers, for example from the manuka tea tree (Leptospermum scoparium) of New Zealand or the jelly bush (Leptospermum polygalifolium) of Australia. Since the first description of UMF, it has been recognized that its concentration is highly variable in different manuka honey batches, and that has been determined to be due to different concentrations of dihydroxyacetone in different cultivars of manuka, with pink-flowered cultivars producing the highest dihydroxyacetone levels in nectar. There are also seasonal changes within a Leptospermum species, or between the different species. Because of this batch to batch variability, the methylglyoxal levels or antibacterial activity of each lot of Leptospermum honey must be assayed to determine whether it will be useful as a medicinal honey or not. As manuka honey often has very low levels of hydrogen peroxide, methylglyoxal becomes its primary antibacterial agent.
A fourth antibacterial factor that has been found in Revamil Source honey that is produced in greenhouses in The Netherlands is Bee Defensin-1, a cationic antimicrobial peptide placed in this honey variety by the bees. Defensins are antimicrobial peptides found in many organisms, including plants, invertebrates, insects, birds and mammals. They are cysteine-rich peptides with multiple disulfide bonds and a triple-stranded beta sheet. Most defensins function by binding to the microbial cell membrane, and once embedded, they form pore-like membrane defects that allow efflux of essential ions and nutrients. Bee Defensin-1, a 51-amino acid peptide (also called Royalisin because it was first discovered in royal jelly), was discovered in Revamil Source honey when bactericidal activity was not eliminated by neutralization of the usual antimicrobial factors (hydrogen peroxide and methylglyoxal). The activity was found in a relatively high molecular weight (>5-kDa) chromatographic fraction; stained as a protein on polyacrylamide gel electrophoresis; and was immuno-stained by anti-bee defensin-1 antibody on a Western blot. In addition, the antibacterial activity of Revamil Source honey was abolished by proteolytic digestion with pepsin and by the anti-bee defensin-1 antibody.
Medicinal honeys from different floral sources exhibit differing antibacterial activities towards different bacterial pathogens. For example, Mundo et al., (2004) reported varying sensitivities to the antibacterial properties of 26 different honey types by nine different bacteria, including multiple strains of Staphylococcus aureus, emphasizing the variability in the antibacterial effect of different honey samples. These authors reported that whereas Bacillus stearothermophilus was the most sensitive microorganism to the antibacterial activity of medicinal honeys in the study, Alcaligenes faecalis, Lactobacillus acidophilus, and Staphylococcus aureus strains ATCC 25923, 8095, and 9144 were each moderately sensitive, and Escherichia coli, Salmonella enterica, Pseudomonas fluorescens, Bacillus cereus, and Listeria monocytogenes were the most resistant to the antibacterial activity of honey.
In the Mundo et al., (2004) study it was demonstrated that different microorganisms had variable susceptibilities to the different antibacterial mechanisms in various honeys. Whereas it required 50% manuka honey with its non-peroxide methylglyoxal antibacterial mechanism to inhibit the growth of B. stearothermophilus, buckwheat honey at only 25% concentration was required to inhibit the growth of this organism via its hydrogen peroxide-dependent antibacterial action. The same was true for the inhibition of S. aureus strains ATCC 25923 and 9144 which both were inhibited by 50% manuka honey but by only 33% buckwheat honey, whereas the converse was true for the inhibition of S. aureus strain ATCC 8095 and B. cereus where 50% buckwheat honey was required to completely inhibit their growth while only 25% manuka honey was required. Table 1 summarizes the bacterial sensitivities of the various bacteria to the different honeys.
TABLE 1Bacterial Sensitivity by Type andInhibitory Concentration of Honey.Type of Honey and (Inhibitory Concentration;Bacteria% honey in water, w/v)E. coli O157:H7christmas berry (100); saw palmetto (100);tarweed (100); buckwheat (100); manuka (50)S. entericamanuka (50)A. faecalisblueberry (100); soybean (100); tarweed (33);buckwheat (33); manuka (25); horsemint (25)P. fluorescenstarweed (100); buckwheat (50)L. acidophilussoybean (100); christmas berry (100); buckwheat(100); manuka (100); saw palmetto (100);melaleuca (50); tarweed (50)L. monocytogenesmelaleuca (100); tarweed (100); buckwheat (100)B. cereustarweed (100); buckwheat (50); manuka (25)S. aureus ATCCchristmas berry (100); saw palmetto (50);8095tarweed (50); buckwheat (50); cotton (33);manuka (25)S. aureus ATCCsaw palmetto (100); sunflower (100); horsemint9144(100); manuka (50); melaleuca (33); buckwheat(33)S. aureus ATCCsoybean (100); sunflower (100); saw palmetto25923(50); melaleuca (50); rabbit bush (50); manuka(50); tarweed (33); buckwheat (33)B. Steorothermophilusblueberry (100); blackberry (100); manuka (50);black sage (50); red sumac (50); melaleuca (50);horsemint (50); christmas berry (50); soybean(33); alfalfa (33); cotton (33); saw palmetto(33); rabbit bush (33); tarweed (25); buckwheat(25); knotweed (20); sunflower (17)
Data from Mundo et al., 2004.
Of the honeys listed in Table 1, buckwheat, tarweed, saw palmetto and melaleuca inhibit bacteria primarily via hydrogen peroxide, whereas the antibacterial activity of manuka, blueberry, and knotweed honeys is primarily non-peroxide mediated. Other studies report similar findings and therefore the present disclosure relates to a honey composition containing high concentrations of both peroxide and non-peroxide antibacterial activities in order to produce a honey with broad-spectrum antibacterial activity efficient at inhibiting the growth of most major wound pathogenic bacteria at one low honey concentration.
As can be understood from the wide variety of compositions, devices and methods directed at wound healing, many strategies have been contemplated to accomplish the desired end. Heretofore, widely administered synthetic organic pharmaceuticals are commonly associated with undesirable side effects and adverse drug interactions. Thus, there is a long-felt need for more natural wound healing compositions. There is further a need for wound healing compositions involving medicinal honey and methylglyoxal, and the corresponding methods of use.